@phdthesis{Kouhestani2021,
  author    = {Kouhestani, Dina},
  title     = {Complementation of a bimolecular Antibody-Derivative within the context of the Immunological Synapse},
  doi       = {10.25972/OPUS-20466},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204669},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Cancer is a disease of uncontrolled cell proliferation and migration. Downregulation of antigen-presenting major histocompatibility complex (MHC) and co-stimulatory molecules are two of the most commonly used pathways by cancer cells to escape from immune surveillance. Therefore, many approaches have been developed for restoring the immune surveillance in cancer patients. One approach is to redirect the patient's own T cells for tumor cell destruction. For T cell function it is important to induce a durable and robust cytotoxic response against target cells and to generate memory T cells, after MHC-mediated recognition of foreign intracellular antigens presented on the surface of antigen presenting cells (APC). Because of these cytotoxic properties, T cell mediated immunotherapy has been established as an effective and durable anti-neoplastic treatment. Different T cell mediated therapies for cancer treatment exist. One of them is using bispecific antibody fragments, so called bi-sepcific T cell engagers (BiTEs), for retargeting of T cells against single antigen positive tumor cells. The BiTE antibodies have two antigen binding domains, one against a target on the target cell, the second against CD3 on the T cells, facilitating cell-to-cell interactions. However, suitable single tumor antigens are limited, which restricts this approach to very few tumor types. To overcome this limitation, we have developed T cell-engaging antibody derivatives, termed hemibodies. Hemibodies exist as two complementary polypeptide chains. Each consists of two specific domains. On one end there is a single-chain variable fragment (scFv) against a target protein and on the other end there is either the heavy chain variable domain (VH) or light chain variable domain (VL) of an anti-CD3 binding antibody. Only when both hemibodies bind their respective antigens on the same tumor cell, the complementary anti CD3 VH and VL domains become aligned and reconstitute the functional CD3 binding-domain to engage T cells. For targeting malignant cells of hematopoietic origin, we used hemibodies against CD45 and HLA-A2. They were expressed in CHO cells, then purified via Strep-tag. To get more insight into the hemibody mechanism of T cell mediated target cell killing, we analyzed the biochemical and functional properties of hemibodies in more detail. Our main finding indicates that VLαCD3-scFvαHLA-A2 and VHαCD3-scFvαCD45 hemibodies induce an atypical immunological synapse characterized by a co-localization of HLA-A2 and CD45 out of the target cell -T cell interface. Nevertheless, hemibodies induce a high caspase activity in target cells in a concentration-dependent manner at nanomolar concentrations in vitro. Looking at ZAP70, which is usually recruited from the cytoplasm to the CD3 receptor in the middle of the cell-cell interface, we were able to detect activated ZAP70 outside of the cell-cell interface in the presence of hemibodies. In contrast cells treated with BiTEs show a central recruitment in the cell-cell interface as expected. We looked also at the interaction of hemibodies with soluble recombinant CD3 epsilon/gamma protein in the absence of target cells. The binding could be measured only at very high concentration out of the therapeutic window. This work contributes to the mechanistic understanding, which underlies the hemibody technology as a new dual antigen restricted T cell-mediated immunotherapy of cancer.},
  language  = {en}
}